Megasphaera elsdenii strain and its uses

ABSTRACT

This invention relates to a novel strain of  Megasphaera elsdenii  and its uses. This invention further relates to preparations and methods incorporating such strain. This invention also relates to feedstuffs for ruminants and a preparation and method for the prevention and treatment of lactic acidosis in ruminants. This invention even further relates to a method of isolating a biologically pure culture of a superior ruminal microorganism in a relatively shorter time period than conventional methods. This invention yet further relates to a method of achieving any one or more of the following improvements in ruminants namely increased milk production; improved feedlot performance; improved growth rate; decrease in finishing time; lower digestive morbidity and mortality; lower incidence of lactic acidosis and related diseases; improved feed conversion efficiency; decrease in roughage content in feeds; and capability to feed on relatively higher concentrate diets.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/521,847, filed Nov. 23, 2005, which is the national stage applicationunder 35 U.S.C. 371 of International Application No. PCT/ZA2003/00092,filed Jul. 15, 2003, which claims the benefit of South African patentapplication No. 02/5743, filed Jul. 18, 2002. The entire contents ofeach of the aforementioned applications are incorporated by referenceherein in their entirety.

INTRODUCTION

This invention relates to a novel strain of Megasphaera elsdenii and itsuses. This invention further relates to preparations and methodsincorporating such strain. This invention also relates to feedstuffs forruminants and a preparation and method for the prevention and treatmentof lactic acidosis in ruminants.

BACKGROUND Lactic Acidosis

Lactic acidosis is a digestive disorder in ruminants that may occur whenthere is a sudden excess intake of readily fermentable carbohydrates,particularly when ruminants are switched from a diet of roughage to ahigh-energy or energy rich concentrate diet containing a high level ofstarch. The disorder is characterized by an accumulation of organicacids, especially lactic acid, in the rumen (Dawson & Allison, 1988).Studies have indicated that a gross imbalance between the numbers oflactic acid-producing bacteria and lactic acid-utilizing bacteria,brought on by a sudden increase in the proportion of readily fermentablecarbohydrates in the diet is the main cause of the onset of lacticacidosis (Slyter, 1976).

Manipulating the rumen microbial population to prevent lactic acidosisby administering material containing high numbers of lactate-utilizingbacteria has been advocated for decades, but never practiced on a largescale. Manipulations to enhance lactate utilization within the rumen hasbeen achieved by administering rumen fluid from an already adaptedanimal (Allison et al., 1964; Braun et al., 1992) and by administeringpure or mixed bacterial cultures of lactate-utilizes (U.S. Pat. No.1,251,483 Wilker et al., 1971; U.S. Pat. No. 3,857,971 Abdo & Chilly,1974; U.S. Pat. No. 4,138, 498 Das, 1979; U.S. Pat. No. 5,380,525 Leedleet al., 1991; Hession & Kung, 1992; Robinson et al., 1992; Wiryawan &Brooker, 1995).

Some of these feed additives containing live bacterial cultures havebeen patented (U.S. Pat. No. 1,251,483 Wilker et al., 1971; U.S. Pat.No. 3,857,971 Abdo & Chilly, 1974; U.S. Pat. No. 4,138,498 Das, 1979;U.S. Pat. No. 5,380,525 Leedle et al., 1991), but not commercializedextensively or at all. In three of the patents (U.S. Pat. No. 1,251,483Wilker et al., 1971; U.S. Pat. No. 3,857,971 Abdo & Chilly, 1974; U.S.Pat. No. 4,138,498 Das, 1979) the cultures were obtained from continuousculture fermenters with an initial inoculum of rumen fluid. However, thedonor animals were not necessarily adapted to a high-concentrate diet.There is also no mention of pH tolerance for any of these cultures. Inthe other patent (U.S. Pat. No. 5,380,525 Leedle et al., 1991) thecultures were isolated at pH 5.3 either directly or indirectly afterenrichment from ruminants adapted to high-concentrate diets.

The Incidence of Sub-acute and Acute Acidosis in Dairy Cattle

Sub-acute rumen acidosis is a common and serious health and productionproblem in the dairy industry because dairy cows are usually fed dietscontaining high levels of grains. Sub-acute and acute rumen acidosis aresimply different degrees of the same problem. Acute rumen acidosis ismore severe and physiological functions may be significantly impaired.The affected animal is depressed and usually ataxic, off-feed, withdilated pupils and an elevated heart rate. Diarrhoea will be obvious andthe animal may become recumbent and die within 2 to 5 days after theinsult (Nordlund, 1995). Acute acidosis is characterized by a dramaticreduction in ruminal pH (≦5.0), a large increase in lactic acidconcentration and a large decrease in protozoa (Nocek, 1997).

Signs of sub-acute rumen acidosis are very-different from that of acuteacidosis. Modern dairy management systems of group housing or groupfeeding make it difficult to recognize these symptoms because individualcows with these problems will usually not be noticed within a group.Herds with sub-acute rumen acidosis will present some or all of thefollowing signs: laminitis, intermittent diarrhoea, poor appetite orcyclical feed intake, high herd cull rates for poorly defined healthproblems, poor body condition in spite of adequate energy intake,abscesses without obvious causes and hemoptysis (coughing of blood) orepistaxis (bleeding from the nose). Most of these signs are secondary toacidosis and most of them do not appear until weeks or months after theinitial acidosis events. Contrary to feedlot cattle, dairy cows are keptfor years and the management of acidosis is therefore of importance inincreasing profits.

Chronic laminitis is perhaps the most consistent clinical sign of a herdwith sub-acute rumen acidosis. Although the relationship betweenacidosis and laminitis is not completely understood, the association iswidely recognized clinically and demonstrated in research trials (Kelly& Leaver, 1990; Manson & Leaver, 1988; Nocek, 1997). Furthermore, mostdairy managers, veterinarians and nutritionists tend to underestimate orperhaps tolerate an abnormal incidence of laminitis and lameness indairy herds. A survey in Minnesota demonstrated a mean incidence inlameness of 15% with a range of 0-33% (Nordlund, Garret & Oetzel, 1995).Studies in Europe have identified lameness as the third most costlyhealth problem in dairy cows after mastitis and reproduction (McDaniel &Wilk, 1989). The management of acidosis is thus clearly of utmostimportance.

A major symptom of sub-acute acidosis is decreased feed intake anddecreased efficiency of milk production. Sub-acute acidosis, because ofdifficulties in diagnosing the problem, tends to be dismissed as otherproblems, such as poor management, poor forage quality e biggesteconomic sink to many dairy farmers because it is omni-present,particularly in high producing dairy herds.

Because of the high incidence of nutritional and metabolic disturbanceamongst high producing dairy cows, nutritional strategies for improvingperformance with cereal based diets focus on the prevention of ruminaldysfunction by controlling acid production or by stimulating moreefficient microbial growth. At present, feed additives play an importantrole in this regard (Hutjens, 1999). The use of yeast culture strainsthat specifically stimulate the growth of lactic acid utilizing bacteriagenerates much interest and a recent survey indicated that yeastcultures are being used in 33% of high producing Wisconsin herds.Results from various studies suggest that the Yea Sacc strain 84170appears to be particularly well suited for altering ruminal fermentationand animal production when used in high lactate silages and feeds highin concentrates (Dawson, 1995). Production results, however, are veryinconsistent. In the USA the cost for yeast culture supplementation is4-6 cents per cow per day (Hutjens, 1999). Ionophores, because of theirability to prevent the growth of important lactic acid producers, canalso play a role in managing sub-acute acidosis. Although the cost isrelatively low (1-2 US cents/cow/day, Hutjens, 1999) there seems to besome resistance against the use of ionophores because of a few recentcases of ionophore toxicity. Furthermore, ionophores have not beenregistered in the USA for use in dairy cattle diets.

Experimentally, there have been several bacteria that have potential asdirect fed microbial (DFM) for ruminants, but have not beencommercialized for a number of reasons. For example, Megasphaeraelsdenii (ME) is the major lactate-utilizing organisms in the rumen ofadapted cattle fed high grain diets. When cattle are shifted from highforage to high concentrate diet, the numbers of ME are ofteninsufficient to prevent lactic acidosis. Kung and Hessian (1995) haveshown that the addition of ME B 159 prevented accumulation of lacticacid during a challenge with highly fermentable carbohydrates. Robinsonet al. (1992) demonstrated that addition of a different strain of ME(407A) prevented lactic acidosis in steers.

Although the costs associated with subclinical ruminal acidosis aredifficult to pinpoint, the potential costs to the dairy industry arehuge (Hall, 1999). Donovan (1997) conservatively estimated the cost ofsubclinical acidosis to the US dairy industry at $500 million to $1billion per year.

Elsden and Lewis (1953) first described a large, strictly anaerobicGram-negative, fatty acid producing, non-motile coccus isolated from therumen of sheep. However, the original isolate was lost before it hadbeen characterized phenotypically in detail. An organism resembling theoriginal strain was isolated from the rumen contents of sheep severalyears later by Elsden and his colleagues (Elsden et al., 1956). Thecharacteristics of this organism did not fit the description of anyknown species at the time, but in view of the small number of isolatesstudied, the authors refrained from assigning the organism to a newspecies and genus, but referred to it as LC. Gutierrez et al. (1959)encountered a similar organism in the rumens of bloating cattle andconcluded that they fell within the definition of the genusPeptostreptococcus, proposing the creation of a new species P. elsdenii.Subsequently, Rogosa (1971) demonstrated that the LC-type isolates wereGram-negative and therefore should not be included in the genusPeptostreptococcus. He proposed transfer of P. elsdenii to a new genusMegasphaera and the new combination M. elsdenii, with the isolate LC1 ofElsden et al. (1956) as the type strain. M. elsdenii is a strictanaerobe found mainly in the rumen of young animals and animalsreceiving high-concentrate diets in which lactate fermentation isparticularly pronounced. The organism has also been isolated on occasionfrom the faeces of humans (Sugihara et al., 1974) and it fermentslactate to mainly butyrate, propionate, isobutyrate, valerat, CO₂, H₂and sometimes trace amounts of caproate (Stewart and Bryant, 1988).Since M. elsdenii is not subject to catabolite repression by glucose ormaltose as in Selenomonas, which is also a lactate utiliser occurring inthe rumen, its contribution to lactate catabolism is particularlyenhanced subsequent to feeding of soluble carbohydrates (Stewart andBryant, 1988).

U.S. Pat. No. 3,956,482 (Hahn et al. 1976) discloses a method ofincreasing milk production in ruminants including the steps ofadministering to the rumen of a lactating cow acetate producingmicro-organisms consisting of a mixture of 0-4% M. elsdenii, 30-42%Streptococcus bovis, 3-10% Lactobacillus acidophilus, 12-20%Bifidobacterium adolescentis, 18-44% Bacteroides ruminicola and 3-12%Butyrivibrio fibrisolvens cultured and adapted to a nutrient medium.

A major disadvantage of the invention disclosed in the above patent isthe relatively high percentage (between 30-42%) of Streptococcus bovis,which together with Lactobacillis is the leading cause of lacticacidosis in ruminants. The mixture further contains a relatively lowpercentage of M. elsdenii (0-4%) and the administration of the mixturewould probably aggravate or initiate ruminal lactic acidosis rather thanpreventing or treating it. The mixture is further exposed to atmosphereso that most of the M. elsdenii perish. A mixture of microorganisms isfurthermore much more difficult to control than a pure culture.

U.S. Pat. No. 4,138,498 (Das, 1979) discloses a feed additive foradministration to ruminants to prevent or minimize lactic acidosis whenruminants are switched from a diet of roughage to starch, comprising abacterial culture of M. elsdenii admixed with an ingestible animal feedadditive. M. elsdenii is strictly anaerobic and a disadvantage of thefeed additive disclosed in this patent, over and above the disadvantagesset out below, is that the M. elsdenii is exposed to atmosphere, leadingto a rapid decline in the amount of viable cells available in theadditive.

U.S. Pat. No. 5,380,525 (Leedle et al., 1991) discloses a biologicallypure culture of M. elsdenii NRRL-18624 and its use in the facilitationof the adaptation of ruminants from a roughage or normal pasture to ahigh-energy starch-rich diet. The culture suffers from the disadvantagesset out below.

U.S. Pat. No. 5,529,793 (Garner et al., 1996) discloses a mixture oflactic acid producing bacteria and a lactate utilizing bacteria such aM. elsdenii with a dry formulation or an animal feedlot diet forimproving the utilization of feedstuffs by a ruminant. A disadvantage ofthis invention is that M. elsdenii is generally strictly anaerobic andthe application thereof to dry feedstuffs would result in most of thecells dying.

The applicants have evaluated the above strains of M. elsdenii and havededucted that they are generally not suitable for commercialization andlarge scale preventative treatment of lactic acidosis in ruminantsbecause of the following disadvantages of these strains namely they arenot:

-   -   highly active and adapted to proliferate in the rumen of animals        on high-concentrate diets;    -   capable of proliferating at relatively low pH values below pH        5.0 and as low as 4.5, characterized as acute acidosis;    -   resistant to ionophore antibiotics commonly added to feedlot        diets; and    -   capable of preferentially using lactate as a carbon source even        in the presence of soluble carbohydrates such as glucose and        maltose.

Further disadvantages of these strains are that, generally, they:

-   -   have a relatively low growth rate, i.e. less than 0.938 h⁻¹;    -   do not have the ability to grow on reducing sugars as well as on        lactate;    -   have a relatively low biomass output rate, i.e. less than 0.39 g        (l. h)⁻¹;    -   are not ionophore resistant; and    -   produce predominantly propionate and butyrate and not        predominantly acetate.

OBJECT OF THE INVENTION

It is therefore an object of the present invention to provide a novelstrain of M. elsdenii and its uses, and preparations and methodsincorporating such strain with which the aforesaid disadvantages can beovercome or at least minimized.

SUMMARY OF THE INVENTION

According to a first aspect of the invention there is provided abiologically pure bacterial culture of M. elsdenii having substantiallythe same 16S ribosomal RNA sequence as that of the M. elsdenii strainCH4 deposited on Mar. 18, 2002 at NCIMB, Aberdeen, Scotland, UK undernumber NCIMB 41125, the M. elsdenii strain CH3 deposited on Nov. 24,2010 at NCIMB. Aberdeen. Scotland, UK under number NCIMB 41787, and theM. elsdenii strain CH7 deposited on Nov. 24, 2010 at NCIMB, Aberdeen,Scotland. UK under number NCIMB 41788. All restrictions imposed by thedepositor on the availability of the deposited material will beirrevocably removed upon the granting of the patent.

According to a second aspect of the invention there is provided abiologically pure bacterial culture of the M. elsdenii strain CH4deposited at NCIMB, Aberdeen, Scotland, UK under number NCIMB 41125, theM. elsdenii strain CH3 deposited on Nov. 24, 2010 at NCIMB. Aberdeen,Scotland, UK under number NCIMB 41787, and the M. elsdenii strain CH7deposited on Nov. 24, 2010 at NCNB, Aberdeen, Scotland, UK under numberNCIMB 41788.

The M. elsdenii strain in accordance with the first and second aspectsof the invention is further characterized by its:

-   -   ability to utilize lactate very efficiently even in the presence        of sugars;    -   resistance to ionophores;    -   relatively high growth rate;    -   capability to produce predominantly acetate; and    -   capability to proliferate at relatively low pH values below 5.0        and as low as 4.5.

According to a third aspect of the invention there is provided acomposition for facilitating the adaptation of ruminants from aroughage-based diet to a high-energy concentrate-based diet, thecomposition consisting essentially of the bacterial culture of the firstor second aspects of the invention.

According to a fourth aspect of the invention there is provided a methodof facilitating the adaptation of ruminants from a roughage-based dietto a high-energy concentrate diet including the step of administering tothe rumen of said ruminants an effective amount of a bacterial cultureaccording to the first or second aspects of the invention.

According to a fifth aspect of the invention there is provided afeed-additive for ruminants comprising a carrier and an effective amountof a bacterial culture according to the first or second aspects of theinvention.

Preferably the culture is disposed in an anaerobic container.

According to a sixth aspect of the invention there is provided a methodfor the treatment of ruminal lactic acidosis and prevention of any oneor more of the following, namely ruminal lactic acidosis, rumenitis,ruminal lactic acidosis induced laminitis, ruminal lactic acidosisinduced bloat and liver abscesses, including the step of anaerobicallyadministering to the rumen of a ruminant an effective amount of abacterial culture according to the first or second aspects of theinvention.

According to a seventh aspect of the invention there is provided aveterinary agent for the treatment of ruminal lactic acidosis andprevention of any one or more of the following, namely ruminal lacticacidosis, rumenitis, ruminal lactic acidosis induced laminitis, ruminallactic acidosis induced bloat and liver abscesses, comprising aneffective amount of a bacterial culture according to the first or secondaspects of the invention.

According to an eighth aspect of the invention there is provided apreparation for the treatment of ruminal lactic acidosis and preventionof any one or more of the following, namely ruminal lactic acidosis,rumenitis, ruminal lactic acidosis induced laminitis, ruminal lacticacidosis induced bloat and liver abscesses in ruminants comprising:

-   -   an inoculum of a bacterial culture according to the first or        second aspects of the invention; and    -   a separate anaerobic growth medium,        the components of the preparation being disposed in separate        chambers of an anaerobic container which are anaerobically        connectable to each other, thus to inoculate the growth medium        with the culture anaerobically.

According to another aspect of the invention there is provided a methodof achieving any one or more of the following improvements in ruminantsnamely:

-   -   increased milk production;    -   improved feedlot performance;    -   improved growth rate;    -   decrease in finishing time;    -   lower digestive morbidity and mortality;    -   lower incidence of lactic acidosis and related diseases;    -   improved feed conversion efficiency; and    -   capability to feed on relatively higher concentrate diets,        including the step of administering to the rumen of a ruminant        an effective amount of a bacterial culture according to the        first or second aspects of the invention.

Preferably the culture is administered anaerobically.

According to yet another aspect of the invention there is provided amethod of isolating a biologically pure culture of a superior ruminalmicroorganism in a relatively shorter time period than conventionalmethods, the method including the steps of:

-   -   obtaining a sample of ruminal fluids; and    -   cultivating the sample on a pre-selected growth medium,        the method being characterized in that a plurality of parameters        selected from the group comprising growth medium constituents,        dilution rate, pH, temperature anti-microbial agents, gaseous        environment, redox potential, lack of nutrients and challenging        organisms, are pre-selected to favor the superior rumen        microorganism to the detriment of inferior rumen microorganisms.

The invention will now be described in more detail below with referenceto the below examples and the enclosed drawings wherein:

FIG. 1 is a graph of growth rates of lactate utilizes at various pHvalues;

FIG. 2 is a graph of the growth rates (h⁻¹) of the lactate utilizingisolates, on glucose medium at various pH values; and

FIG. 3 is the phylogenetic tree of M. elsdenii according to the presentinvention.

In accordance with the present invention, organisms capable of utilizinglactic acid were isolated directly from ruminants adapted to ahigh-concentrate diet. The objective was to select those cultures withthe best combination of characteristics for the purpose of applicationas mass-cultured, preserved inocula for prophylactic and/or therapeutictreatment of lactic acidosis.

For the lactate-utilizing bacteria to be effective they must be highlyactive and adapted to multiplication in the rumen of animals onhigh-concentrate diets. The organisms should be able to multiply at pHvalues below pH 5.0. The selected isolates should also be resistant toionophore antibiotics commonly added to feedlot diets. Lactate should bepreferentially used as a carbon source even in the presence of solublecarbohydrates such as glucose and maltose, which would be present inhigh proportions in high-concentrate diets.

METHODS

1. Animals Used During Isolations

Samples of rumen contents from animals with a pre-selection for lactateutilizing bacteria were chosen, namely lactating fistulated dairy cowsat the Dairy Cow Nutrition unit, Irene, of the Agricultural ResearchCouncil (South Africa), as well as feedlot cattle of Chalmar Beef(Pretoria, South Africa) which were slaughtered at the end of theirfinishing periods. All the animals were adapted to high-concentratediets, which increased the numbers of naturally occurring lactateutilizing bacteria.

2. Sample Collection and Preparation

Samples of rumen contents were collected from dairy cows at about 09h00,after the cows had been fed and milked. Samples of rumen contents fromfeedlot animals were obtained 15-30 minutes after the animals had beenslaughtered. Plastic screw-cap sample bottles were filled to capacitywith rumen fluid filtered through two layers of cheesecloth. The rumenfluid was transferred directly into the fermenter.

3. pH-auxostat

A New Brunswick Scientific Bioflo 1 continuous culture system wasmodified into a pH-auxostat by converting the pH-dosing pump to a mediumaddition pump. The pH was monitored with a Schott S23158 pH-electrodeconnected to a Digital Data Systems 302 pH-meter and titrator. A poorlybuffered medium was added whenever the pH increased over the set valueuntil the desired value was reached. The working volume of the culturevessel was 270 ml. The maximum dilution rate obtained for a givenorganism during auxostat cultivation is a measure of the maximum growthrate of that organism during that condition.

4. Isolation of Lactate Utilizing Rumen Bacteria via the Auxostat

4.1 Growth Conditions and Medium

Filtered rumen fluid was used to fill the fermenter (270 ml) initiallyand the titrator activated to add sterile medium (Medium 1) to theculture proportionally to the increase in pH of the culture. Medium 1was a semi-defined rumen fluid free medium consisting of: Na-lactate(70%), 10 g/l; Peptone, 2 g/l; KH₂PO₄ 1 g/l; (NH₄)₂SO₄ 3 g/l; MgSO₄ 7H2O0.2 g/l; CaCl₂.2H₂O 0.06 g/l; Vitamins (Pyridoxolhydrochloride, 4 mg/l;Pyridoxamine, 4 mg/l; Riboflavin, 4 mg/l; Thiaminiumchloride, 4 mg/l;Nicotinamide, 4 mg/l; Ca-D-pantothenate, 4 mg/l; 4-Aminobenzoic acid,0.2 mg/l, Biotin, 0.2 mg/l, Folic acid, 0.1 mg/l and Cyanocobalamin,0.02 mg/1); Na₂S.9H₂O, 0.25 g/l; Cysteine, 0.25 g/l; Antifoam, 0.07 ml/land Monensin, 10 mg/l. The Na-lactate and mineral solution were bothadded to the reservoir boffle and autoclaved for 60 min. The peptone wasdissolved in 300 ml d.H₂O and autoclaved separately in a 1.0 | Schottbottle with a bottom outlet fitted with Quick-fit glass connections. Thevitamin solution was filter sterilized beforehand as well as the tworeducing agents. Following autoclaving, the reservoir bottle was gassedwith anaerobic gas overnight and the other constituents added separatelyafter cooling. The pH was adjusted to the desired value with 5N HCl.

Continuous culturing followed until a pure culture was observedmicroscopically. A sample was taken from the fermenter with a sterilesyringe, which was sealed and transferred into the anaerobic cabinet(Forma Scientific model 1024). One droplet of the culture was streakedout in a Petri dish containing Medium 1 solidified with 2% agar.Incubation followed at 39° C. overnight and a single colony wastransferred with a sterile needle and syringe into a fresh Medium 1contained in a 30 ml serum bottle. After incubation at 39° C. for 24 hthe culture was transferred to several slants containing Medium 1 andincubated overnight. These slants were stored above liquid nitrogen forlong-term preservation.

4.2 Batch Growth Rates of Isolates in Fermenter

The growth rates of the isolates were verified using the batchcultivation technique and monitoring the increase in optical densityover time. The natural log of the optical density (OD) was plottedagainst time and the linear part of the graph was used to determine theslope, which represented the maximum growth rate of the organism.Determination of the batch growth rate was performed in a chemostatculture, which was diluted with a sterile medium until a very diluteculture suspension was obtained and the medium supply cut off in orderto start the batch growth. The advantage of using a chemostat culturefor this work is that there is no lag phase since the cells are allviable and adapted to the medium.

4.3 Analytical Techniques

Volatile fatty acids were determined by gas chromatography with a CarloErba GC4200 gas chromatograph with flame ionization detector and aTupelo 1-1825 column (Supelco Inc., Bellefonte Pa., USA). Operatingconditions were as follows: carrier gas, nitrogen; flame gases, hydrogenand air; column temperature 175° C.; injection port temperature 200° C.A Barspec data system (Barspec Systems Inc., Rehovot, Israel) was usedfor peak integration. Pivalic acid served as the internal standard. Theutilization of the D-and L-lactate isomers were determined enzymatically(Test combination 1112 821, Boehringer Mannheim GmbH, Mannheim).

5. Isolations of Bacteria via Spread Plate Method

5.1 Culture Media

The incubated rumen fluid lactate (IRFL) medium for the spread plateisolations consisted of 400 ml incubated clarified rumen fluid (Olumeyanet al., 1986) from lucerne-fed sheep, 371 ml distilled water, 2 gpeptone (Merck), 15 g agar, 100 ml 10% (w/v) sodium-D, L-lactatesolution, 100 ml 0.04% (w/v) bromocresol purple solution and 25 mlmineral solution containing 40 g/l KH₂PO₄; 120 g/l (NH₄)₂SO₄; 8 g/lMgSO₄.7H₂O and 2.4 g/l CaCl₂.2H₂O. Lactic acid (90% w/v) was used toadjust the pH to 5.5 before autoclaving at 121° C. for 25 minutes. Aftersterilization the medium was cooled down in a 50° C. water bath whilebeing gassed with an anaerobic gas mixture. Two milliliters of each ofthe reducing agents, Na₂S.9H₂O (12.5% w/v) and cysteine.HCl.H₂O (12.5%w/v) were added aseptically. As IRFL medium is not completely selectivefor lactate-utilizes, bromocresol purple was incorporated to facilitatedetection of lactate-utilizes. When lactate is utilized there is achange in ionic balance in the immediate vicinity of the colony causinga pH increase. A rise in pH above 6.3 was indicated as a color changefrom yellow to purple in the zone concentric with the culture.

Acid tolerance was determined on IRFL agar plates with initial medium pHvalues of 4.5, 5.0 and 5.5.

Resistance to ionophores was tested on IRFL agar plates containing 10ppm of ionophores generally used in high-concentrate diets i.e. monensin(Sigma) and lasalocid (Sigma). Repression of lactate utilization bysoluble sugars was tested on IRFL agar plates supplemented with maltoseor glucose at a final concentration of 10 g/l. A positive result i.e. apurple zone concentric with the colony indicated that the rate of baserelease due to lactate utilization exceeded that of acid production fromthe added sugar. The isolates were also screened on IRFL agar mediumwithout lactate, but to which glucose or maltose had been added at aconcentration of 10 g/l, to determine utilization of the two sugars.

Growth rates on maltose and glucose were determined on media similar toSDL medium, but in which the lactate was replaced with either 10 g/lglucose or maltose.

5.2 Spread Plate Isolations and Screening

The samples for the spread plate isolations were diluted (Mackie &Heath, 1979) in an anaerobic cabinet. Spread plates of IRFL medium wereprepared with the 10⁻⁴ to 10⁻⁶ dilutions and incubated anaerobically at39° C. After 24 hours, well-spaced colonies showing a purple zone weretransferred to IRFL liquid medium in 1.5 ml microtubes. The inoculatedmicrotubes, which showed a color change to purple within 16 hours, werescreened for acid tolerance, ionophore resistance, catabolite repressionand utilization of glucose and/or maltose. Screening was done by replicaplating (Lederberg & Lederberg, 1952) using a multipoint inoculator toinoculate 20 isolates onto a set of nine agar plates of differentcompositions described above.

5.3 Growth Rate Determinations

Growth was measured, in triplicate, in SDL, SDG or SDM medium as anincrease in turbidity at 578 nm. Vials were incubated in a water bath at39° C. between readings. Readings were continued until the turbidityreached the limit of a satisfactory relationship with biomass. Thenatural logarithms of optical density (OD) were plotted againstincubation time. The slope of the exponential growth phase thatrepresents the specific growth rate was calculated by linear regressionwith the aid of a spreadsheet software package.

The cultures grown on SDL medium at pH 5.7 were then incubated furtherfor a total of 24 hours after which 9 ml was preserved by the additionof one milliliter 10% (w/v) NaOH for analysis of end-products formed andutilization of lactate isomers.

5.4 Growth Physiological Studies of Plate Isolates

Fermenter description. A continuous culture system was set up with threefermenters with a capacity of about 250 ml each. A single peristalticpump was used to supply medium at different rates to the threefermenters. Temperature of the cultures was maintained at 39° C. Themedium and fermenters were gassed with 100% CO₂ to maintain an anaerobicenvironment. The pH of the cultures was maintained at pH 5.5 by additionof 20% (w/v) orthophosphoric acid as required. The dilution rate was setat 70%, 80% and 90% of maximum growth rate. A sample of 80 ml waswithdrawn aseptically from each fermenter at steady state. From thissample the dry mass of the cells and the residual lactic acid in themedium was determined. The biomass output rate, a product of thedilution rate and steady-state biomass, was calculated using the actualdilution rate and the dry mass figures. The growth yield coefficient,which is a function of the biomass concentration at steady state overthe amount of substrate utilized, was calculated using lactic acidresidue and dry mass figures.

6. Trials with Sheep to Evaluate the Ability of Isolate CH4 to PreventRuminal Lactic Acid Accumulation

6.1 Methods

In the first trial 12 ruminally-cannulated wether sheep (mean liveweight ca 40 kg) were randomly divided into a treatment and a controlgroup, each comprising six animals. All animals were fed roughage ad libfor 21 days. On day 21 they were fasted for 11 hours prior to beingoffered 1000 g of maize meal/animal and at the same time being dosedintra-ruminally with 300 g of maltose syrup/animal. One hour later allmaize not yet consumed by each animal was packed directly into itsrumen. Immediately thereafter animals in the treatment group were dosedintra-ruminally with 1×10¹¹ cfu of CH4, whilst animals in the controlgroup were similarly dosed with cell-free filtrate of CH4 preparation,i.e. CH4-free. Samples of rumen fluid were taken at two-hourlyintervals, up to 12 h post dosing, for determination of rumen lacticacid concentration.

In the second trial another group of 12 ruminally-cannulated wethers,mean live weight 29 kg, with no previous exposure to concentratefeeding, were used.

They had random access to ground Eragrostis teff hay and aprotein-mineral lick. Lambs were randomly divided into two groups of sixanimals each, namely a treatment and a control group. On the first dayof the experiment (Day 1) all lambs received the following diet ad lib:maize, 888; molasses, 69; urea, 17; limestone, 11; dicalcium phosphate,6; salt, 4; ammonium sulphate, 4; mineral-vitamin premix with monensin,1 (g/kg DM). On day one of concentrate feeding each animal in thetreatment group received a dose of CH4 intra-ruminal at 12:00, i.e. 3hpost feeding. Animals in the control group were similarly dosed withwater. Rumen samples were obtained at various times on the day prior tothe start of concentrate feeding (Day-1) and on the 1^(st), 2^(nd),3^(rd) and 7^(th) days of concentrate feeding, for determination ofruminal lactic acid concentrations.

7. Evaluation of Isolate CH4 in High Producing Dairy Cows

7.1 Cultivation of Lactate Utiliser for the Animal Trial

A Braun Biostat B fermenter with a working volume of 10 liters wastransformed into a chemostat using a Watson-Marlow 505S dosing pumpequipped with a 55 rpm drive for transporting sterile medium from 50liter stainless steel kegs. The working volume was kept constant bycontinuously transferring excess of culture above the 10-liter level ofthe fermenter via a dip tube and a peristaltic pump (Watson-Marlow 505S)to a 50 liter polypropylene Carboy that was cooled in a chest freezer.The delivery rate of this harvest pump was set to approximately 120% ofthe medium supply pump. The excess of volume removed from the fermenterconsisted of anaerobic gas from the headspace.

A tangential flow filter system (Millipore Pellicon) equipped with aMillipore HVMP 0.45 micrometer (15 ft²) filter and a MilliporeMasterflex Easy-Load peristaltic pump, was used for concentrating theculture.

The medium used was CSL4. The vitamin, reducing agent, mineral and traceelement solutions were filter sterilized prior to addition to the mediumreservoir. Following autoclaving, the reservoir was gassed withanaerobic gas.

The production approach was a staggered type of production. Twoconsecutive productions were performed, each producing enough cells forthe treatment of one day's group of animals. The number of concentrationsteps was limited to one per production as each day's production wascollected into a 50 liter vessel. The dilution rate of the culture was0.4 h⁻¹ and the “down time” between batches was 50 minutes. A backup runconsisting of 45 liters was started prior to the first day's production,which also served to promote the chemostat culture into steady state.

7.2 Experimental Animals

Sixty high producing dairy cows were blocked according to milkproduction during previous lactation and body weight and thereafterrandomly allocated, within each block, to one of the followingtreatments: 1) Control diet 60% concentrate; 2) Control diet 60%concentrate+CH4; 3) Control diet 70% concentrate; 4) Control diet 70%concentrate+CH4. Cows were dosed with organism CH4 at calving, 10 dayspost partum and 20 days post partum.

The following parameters were monitored:

-   -   1. dry matter intake daily;    -   2. milk production daily;    -   3. milk fat, protein and lactose weekly; and    -   4. body weight and condition score monthly.        8. Statistical Analyses

Data were analyzed by analysis of variance for a completely randomizedblock design using the program Genstat 5. Previous lactation milkproductions were used as a covariate and milk production was reported ascovariate adjusted values. Contrasts were used to determine thesignificance of difference among treatments as follows:

-   -   +CH4 or −CH4 (dosed vs. non-dosed)    -   Control diet 70% concentrate vs. control diet 70% concentrate        +CH4    -   Control diet 60% concentrate vs. control diet 60% concentrate        +CH4

Differences were declared significant at P<0.10 and trends were declaredat P<0.15 unless otherwise noted.

9. Effects of Megasphaera elsdenii Supplementation on Animal Health andFeedlot Performance

A homogenous group of 448 Bonsmara weaner steers (average initial liveweight 215 kg) were randomly assigned to eight experimental treatmentsin a 2×2×2 factorial design, with factors (1) CH4 addition (yes or no);(2) ionophore addition (yes or no); and (3) roughage level (high orlow). The feedlot diets used and the dietary regime followed were asfollows:

Ingredient composition of experimental diets, at the end of theadaptation period (day 14 to end):

Inclusion (%, on as-fed basis) Ingredient High-roughage Low-roughageEragrostis hay 8.0 2.0 Maize meal 27.0 30.0 Hominy chop 32.0 35.0Molasses meal 12.0 12.0 Brewers grain 6.0 6.0 Wheat bran 10.0 10.0Cottonseed oilcake 2.0 2.0 Urea 1.0 1.0 Limestone 1.5 1.5 Salt 0.5 0.5

Dietary adaptation regime followed from arrival, when animals were givenroughage ad lib, until they were on the final feedlot diet:

Roughage level (%) in the diet Day Additional hay High-roughageLow-roughage 1-2 Ad lib 18 12 3-4 None 18 12 5-7 None 14 8  8-10 None 126 11-13 None 10 4 14 to end None 8 2

Nutrient composition of the final feedlot diets used (% of as-fed):

Nutrient High-roughage Low-roughage Dry matter 89.6 89.8 Crude protein14.4 13.4 Starch 33.5 35.9 NDF fibre 26.9 23.9 ADF fibre 9.5 8.7 Fat 4.64.8

Animals were kept in small experimental feedlot pens. There were 7 pensper treatment and 8 animals per pen. Feedlot diets were fed once dailyin the morning, at an ad-lib level. All steers were processed uponarrival (standard feedlot procedures) and fed only long roughage for afew days until they were dosed with either CH4 (treatment) or a similaramount of water (controls). During this dosing a 200 ml suspension ofCH4 in medium was applied as an once-off oral drench to each treatmentanimal.

The CH4 culture was prepared by inoculating a 17.5-liter batch ofsterile CSL6 medium (starting pH of 5.20) with 1000 ml of fresh inoculumof CH4, pumped directly from fermenter to Carboy container and incubatedat 39° C. overnight. The pH of the culture was 6.63 after cultivationand remained the same after 48 hrs. Counts were done on the CH4 culture,after incubation. A peristaltic pump was used to transfer the 200 mldosage per os to the animal in 10 seconds from the 20-liter carboy.

CSL 6 medium as a 20 liter batch for cultivation of CH4:

17.5 Litres CSL 6 Medium (Sterilized: 55 minutes) Na-lactate 971.25 gIndigocarmine 17.5 ml Trace Mineral solution 8.75 ml Mineral solution 587.5 ml Peptone 17.5 g Yeast extract 17.5 g CSL 598.5 g Distilled H₂010688.2 g 10 N KOH 58.3 ml (pre-dissolved in 5 l H₂0) Filter sterilizeand added prior to inoculation Vitamin solution 35 ml L-cysteine 35 ml

Feed intake was determined for each pen (daily/weekly) and individualanimal weights were determined (weekly/bi-weekly). These were used tocalculate feed conversion ratio (per pen). Animals were observed dailyand any animals showing signs of acidosis (diarrhoea, bloating,depression) were removed and treated before returning them to theirrespective pens right away.

Statistical Analyses

Data were analyzed using the program GenStat 5. Animals were blocked byweight group. The effects of CH4, ionophore and roughage level weretested by means of a 2×2×2 factorial design in an analysis of variance(ANOVA). The data was acceptably normal with homogenous treatmentvariances. Treatment means were separated using Fishers' protectedt-test least significant difference (LSD) at the 5% level, provided thatthe F-probability from the ANOVA was significant at 5%.

10. Identification of Isolates using Phylogenetics, Based on 16S rRNAGene Sequences.

10.1 Bacterial Isolates and Culture Conditions.

M. elsdenii isolates CH4 and CH7, originally isolated from dairy cows(Wiederhold, 1994) were provided by the inventors. The type strain of M.elsdenii, ATCC 25940, was obtained from the American Type CultureCollection. The strains were cultivated in SDL medium as describedpreviously and presumptively identified as Megasphaera elsdenii(Wiederhold, 1994).

10.2 Amplification and Sequencing of 16S ribosomal RNA Genes.

Genomic DNA was extracted from bacterial cells using standard procedures(Ausubel et al., 1988). The primers used to amplify the 16S rRNA geneswere selected from universally conserved regions in all eubacteria(Table 1). PCR was carried out using primers FD1 (covering positions 8to 26) and R11 (positions 1384-1400). All target positions of primersused for amplification and sequencing refer to the E. coli numberingsystem (Brosius et al., 1978). The PCR reaction mixture of 100 μlcontained approximately 200 ng of DNA, 1 μM of each primer, 200 μM ofeach nucleotide (dATP, dCTP, dGTP and dTTP), 50 mM KCl, 10 mM Tris-HCl(pH 8.4) and 2.5 mM MgCl and 2.5 U of Taq polymerase (BoehringerMannheim, Germany). The mixture was overlaid with liquid paraffin toprevent evaporation. The thermal profile consisted of 30 cycles ofdenaturation for 1 minute at 94° C., annealing at 45° C. for 2 minutesand subsequent extension at 72° C. for 3 minutes in a thermal cycler(Hybaid, U.K). Final extension was carried out at 72° C. for 6 minutes.The homogeneity of the amplicons was analyzed by agarose gelelectrophoresis (Sambrook et al., 1989). The PCR product was excisedfrom the gel and purified using the Wizard PCR Preps kit (Promega,U.S.A.) as prescribed by the manufacturer. Direct sequencing of doublestranded PCR amplicons and subsequent separation of sequencing reactionproducts on polyacrylamide gels were essentially carried out accordingto the protocol of Dorsch and Stackebrandt (1992). Sequencing primersare listed in Table 1.

TABLE 1 Primers used to amplify and sequence the 16S rRNAgene. Primer sequences have been publishedpreviously (Dorsch and Stackebrandt, 1992; Lane etal., 1985; Stackebrandt and Charfreitag, 1990;Hutson et al., 1993). A combination of theseprimers covered a total of 1419 nucleotides of the 16S rRNA gene.Primer Target position^(a) Primer Target Primer Target Primerposition^(a) position^(a) Sequence Primer sequence Primer sequence (5′to 3′) (5′ to 3′) (5′ to 3′) Reverse direction (antisense) R11 (PCR)1384-1400 CGGTGTGTACAAGGCCC [SEQ ID: NO: 1] R1193 1174-1192CGTCATCCCCGCCTTCCTC [SEQ ID: NO: 2] R1353 1336-1352 CGATTACTAGCGATTCC[SEQ ID: NO: 3] R961/R7 949-963 TCGAATTAAACCACA [SEQ ID: NO: 4] R5786-802 CTACCAGGGTATCTAAT [SEQ ID: NO: 5] R361/R1 340-355CTGCTGCCTCCCGTAGG [SEQ ID: NO: 6] Forward direction (sense) FD1/F1 (PCR) 8-26 AGAGTTTGATCCTGGCTCA [SEQ ID: NO: 7] F1353 1336-1352GGAATCGCTAGTAATCG [SEQ ID: NO: 8] F361 340-355 CCTACGGGAGGCAGCAG[SEQ ID: NO: 9] F961 949-963 TGTGGTTTAATTCGA [SEQ ID: NO: 10] ^(a)Alltarget positions for primers refer to E. coli numbering systems (Brosiuset al., 1978).10.3 Data Analysis.

The 16S rDNA sequences obtained were automatically aligned withsequences obtained from the Ribosomal Database Project (RDP; Maidak etal., 1996) using the alignment program CLUSTALW (Genetics ComputerGroup, 1991). Sequences in the profile were trimmed in order tostandardize with regard to the size of the sequences of each organismincluded in the alignment profile. A total of 1388 nucleotide sequencepositions were included in the profile. Published sequences of a numberof organisms occurring in the rumen were included in the alignmentprofile (Table 2). Ambiguous sequences in the alignment profile weremanually aligned using the Genetics Data Environment (GDE) alignmenteditor (Smith, 1992). For inferring phylogenetic relationships, theprogram fastDNAml (Olsen et al., 1994) was used, which is based on themaximum likelihood algorithm (Felsenstein, 1981). A phylogenetic treewas constructed using the program Treetool (GDE). Escherichia coli andAcinetobacter calcoaceticus served as out groups in the construction ofthe tree.

TABLE 2 Organisms included in the alignment profile using the programsCLUSTALW. All the sequences were retrieved from the RDP and the Genbankdatabases. Lactobacillus ruminis ATCC 27780 Streptococcus bovis ATCC33317 Fibrobacter succinogenes S85 ATCC 1916 Methanobrevibacterruminantium ATCC 35063 Megasphaera elsdenii ATCC 17752 Methanobacteriumformicicum DSM 1312 M. elsdenii ATCC 25940 Methanosarcina barkeri DSM1538 M. elsdenii CH4 Methanomicrobium mobile ATC 35094 M. elsdenii CH7Prevotella ruminicola ATCC 19189 M. cerevisiae Wolinella succinogenesATCC 33913 Synergistes jonesii Escherichia coli Clostridiumacetobutylicum ATCC 824 Acinetobacter calcoaceticus ATCC 33604Eubacterium cellulosolvens ATCC 43171 Quinella ovalis Eubacteriumuniformis ATCC 35992 Selenomonas ruminantium GA192 Clostridiumpolysaccharolyticum ATCC 33142 Eubacterium limosum ATCC 8486

RESULTS

Auxostat Isolations

Isolation of Lactate Utilizing Bacteria in Auxostat

Isolation 1: The rumen contents, obtained from cow 8710, filling theculture vessel of the fermenter were immediately exposed to freshsterile selective medium when the auxostat was triggered by an increasein pH. Initial dilution rates were in the region of 0.53 h⁻¹ for thefirst 2 hours at pH 5.30. During the following two hours the dilutionrate increased to 0.65 h⁻¹. In order to increase the specificity of theisolation the pH was decreased to pH 5.0, which resulted in a decreasein dilution rate to 0.37 h⁻¹. Cultivation was continued for a further 24hours after which time only two morphological types could be detected inthe enrichment culture. The dilution rate decreased slightly as thecultivation time increased after the initial 24-hour period and at theend of the isolation the dilution rate was only 0.33 h⁻¹.

A sample of the fermenter contents was streaked onto agar medium, in theanaerobic cabinet, and a single colony containing a pure culture wastransferred to agar slants and preserved over liquid nitrogen and thisculture was denoted as Isolate CH1.

Isolation 2: During this isolation from rumen contents of cow 8812 adilution rate of 0.25 h⁻¹ was observed for the first 24 hours and duringthe subsequent 24-hour period the dilution rate was between 0.34 and0.41 h⁻¹. After 48 hours of cultivation it was not clear whether a“pure” culture had been obtained and the cultivation proceeded for afurther 24 hours. The dilution rate during this period was 0.41 h⁻¹ anda pure culture was isolated from the fermenter via a colony from a Petridish. This isolate was designated Isolate CH2.

Isolation 3: The dilution rate during this isolation period decreasedfrom, 0.28 to 0.21 h⁻¹ over a 48-hour period. The isolate obtained fromthe rumen of cow 8708 was designated Isolate CH3.

Isolation 4: Initial dilution rates were in the order of 0.38 h⁻¹, butwithin 4 hours the dilution rate decreased to 0.276 h⁻¹ and at the endof the 48-hour period the dilution was only 0.197 h⁻¹. The isolateobtained during this isolation from the rumen content of cow 8826 wasdesignated CH4.

Isolation 5: At the end of the isolation period a spore former was thedominant organism and the experiment was terminated.

Isolation 6: During this isolation the dilution rate decrease followingthe same pattern as for the other isolations and the final dilution ratewas only 0.116 h⁻¹. The isolate was obtained from rumen contents offeedlot cattle and was designated CH6.

Isolation 7: The rumen contents used during this isolation were obtainedfrom feedlot cattle. The dilution rates decreased from 0.142 to 0.106h⁻¹, during the first seven hours of isolation. The isolate obtained wasdesignated CH7.

Medium Modification for Chemostat Studies

A consistent decrease in dilution rates was observed during theisolation of the lactate utilizes, which indicated that the formulationof the medium was not optimal. During the first 24 hours of isolation 7the dilution rate decreased from 0.142 to 0.106 h⁻¹. A pulse dose of 5ml sterile rumen fluid was added directly into the fermenter and after 4hours the dilution rate peaked at 0.408 h⁻¹. Thereafter the dilutionrate slowly decreased to 0.15 h⁻¹. This “pulse and shift” techniquedemonstrated that the medium was nutritionally deficient.

Another “pulse and shift” experiment with 1 ml vitamin solution resultedin a dilution rate peak of only 0.28 h⁻¹. However, a larger vitaminpulse resulted in a dilution rate peak of 0.497 h⁻¹, which was higherthan with the rumen contents pulse. Lactate utilization reflected thesame results namely, respective b-and L-lactate isomer utilization of 22and 86% without extra vitamins, and with extra vitamins respectively 68and 91%. Medium 1 listed in the Methods reflect the modified versionwith higher concentrations of the vitamins. During another “pulse andshift” experiment it was established that yeast extract increased thecell yields of the isolates.

Growth Rates of Megasphaera elsdenii ATCC 25940 and auxostat isolatesvs. pH

Growth rates of the bacteria were determined with the pH-auxostat atvarious pH values between 4.5 and 6.5, using the modified lactatemedium. These growth rates were checked against the values obtainedduring batch cultivation at the specific pH values and the average valuewas used.

Megasphaera elsdenii ATCC 25940, the type strain, showed an increase ingrowth rate from pH 4.5 up to pH 6.0, followed by a rapid decrease ingrowth rate at pH 6.5 (FIG. 1). The maximum growth rate achieved by ATCC25940 was 0.66 h⁻¹, which corresponds to the reported growth rate of 0.6h⁻¹ by Therion et al. (1981).

All the isolates outperformed ATCC 25940 as far as maximum growth ratewas concerned, especially at pH values of 5.5 and below (FIG. 1). Themaximum growth rates of the isolates all peaked at pH 5.5 withrespective growth rates, (h⁻¹) of 0.66, 0.93, 0.938 and 0.864 forisolates CH7, CH6, CH4 and CH3. Of all the isolates, CH4 proved to bethe most acid tolerant with a growth rate of 0.389 h⁻¹ at pH 4.5, andthe organism with the second best acid tolerance, CH6, with a growthrate of only 0.19 h⁻¹ at pH 4.5. A sharp decrease in growth rate betweenpH 5.5 and 6.0 was observed for three isolates, namely CH6, CH4 and CH3.Isolate CH7 had only a slight variation in growth rate between pH 5.0and 6.0, which resembles ATCC 25940 between pH 5.5 and 6.5.

Growth Rates of Auxostat Isolates on Glucose

The growth rates of three isolates were determined at pH 5.0, 5.5 and6.0, using the fed-batch growth technique (FIG. 2). Growth rates werenoticeably lower for all three isolates on glucose compared to lactate.The most promising isolate on lactate, namely CH4 achieved a maximumgrowth rate of only 0.25 h⁻¹ at pH 5.5 on glucose, compared to 0.938 h⁻¹on lactate. Isolate CH7 achieved the highest growth rate (0.33 h⁻¹) onglucose at pH 6.0 amongst the isolates.

Conversion of Lactate by Isolate CH4

Isolate CH4 was cultivated at three chemostat dilution rates, namely0.94, 0.83 and 0.75 h⁻¹ on lactate medium. During steady state, sampleswere taken and analyzed for volatile fatty acids (VFA) and theutilization of lactate determined. Batch cultivation was also conductedand samples were taken at stationary phase. Samples of the sterilemedium were also analyzed for VFAs and lactate.

With an increase in dilution rate the relative production of fatty acidschanged, namely at low dilution rates more butyrate and valerat wereproduced and less propionate and acetate (Table 3). At the highestdilution rate very small amounts of butyrate were produced, with novalerat and only slightly more acetate and propionate. Lactateutilization decreased as expected, with an increase in dilution rate.During the cultivation D=0.75 more than 40% of the lactate was convertedto VFAs and although lactate utilization was high, a large proportion ofthe available energy was wasted. When CH4 was cultivated in batch itproduced mainly acetate and propionate. The concentrations of VFAsproduced during batch cultivation were much lower than expected and theonly explanation would be that CH4 utilizes the VFAs when lactate isdepleted.

TABLE 3 Volatile fatty acids produced by isolate CH4 from lactate duringchemostat cultivation at various dilution rates and during batchcultivation. Dilution Volatile fatty acids (mM) % Lactate rate (h⁻¹)Acetic Propionic n-Butyric n-Valeric utilised 0.75 7.221 5.779 11.3476.383 92.66 0.83 10.048 12.293 0.423 0.012 53.54 0.94 8.529 10.517 0.2710 39.65 Batch 10.659 7.737 0.266 0 97.62Spread Plate Isolations and Screening

More than 800 colonies from nine rumen samples of four dairy cattle andtwo feedlot cattle were inoculated into IRFL liquid medium inmicrotubes. Of these 610 produced a color change to purple in the mediumwithin 16 hours of incubation. Nineteen of the screened isolates werechosen for further characterization as they met the specificationsrequired.

Four of the selected isolates, AW09; AW10; AW11 and AW12 were capable ofgrowth at an initial pH of 4.5. The other fifteen isolates all grew atan initial pH of 5.0 and there was further selection here, as thosecultures showing suitable characteristics, being the fastest growers atpH 5.0, were selected.

All nineteen isolates were resistant to the ionospheres monensin andlasalocid at concentrations of 10 ppm, utilized lactate in the presenceof maltose and glucose and were capable of growth on both glucose andmaltose. These nineteen isolates were all Gram-negative cocci (±1.8micrometers) occurring in pairs or chains.

Physiological Characterization of Isolates

All the AW isolates used both the D-and L-lactate isomers as bothisomers were virtually completely utilized after incubation in SDLmedium for 24 hours. Results indicated that the isolates comprised afairly uniform group and therefore only certain isolates were chosen forfurther characterization. For five of the AW isolates the growth rateson glucose at pH 5.8 ranged from 0.38 to 1.05 h⁻¹ with a mean of 0.66 h⁻(+/−0.298). The AW isolates tested for VFA production from DL-lactatewere found to produce acetic, propionic, n-butyric and n-valeric acidsin the following ratio 2:1.5:1:1.3. Some of the AW isolates producedtrace amounts of methyl butyric acid. The maximum biomass output ratesobtained for the nine isolates ranged from 0.31 to 0.43 g (l.h)⁻. AW15had the highest biomass output rate and CH4 and AW01 were next in linewith 0.39 g (l.h)⁻¹. The yield of cell dry mass per gram of lactic acidutilized, ranged from 0.1 to 0.17 for the nine isolates.

Presumptive Identification of Isolates

The isolates obtained were presumptively identified and those meetingthe morphological typing as strains of Megasphaera elsdenii were usedfor further characterization.

Trials with Sheep to Evaluate the Ability of Isolate CH4 to PreventRuminal Lactic Acid Accumulation

Results of the first sheep trial are shown in Table 4.

TABLE 4 Lactic acid concentration in rumen fluid of roughage-fed sheepsuddenly challenged with concentrates, and being dosed intra-ruminallywith CH4 (treatment) or placebo (control) at the same time. Time afterLactic acid concentration (g/liter) CH4 dosing (h) CH4 treatment Control0 <0.1 <0.1 2 0.3 1.4 6 0.8 3.6 8 0.5 5.2 10 0.4 6.1 12 0.2 5.9

Results of the second sheep trial are shown in Table 5.

TABLE 5 Lactic acid concentration in rumen fluid of roughage-fed sheepsuddenly changed onto a concentrate diet (ad lib), and being dosedintra-ruminally with CH4 (treatment) or placebo (control) 3 h afterfirst receiving the concentrate diet. Day of Ruminal lactic acidconcentration (mMol/liter) treatment Time of day: 08:00 12:00 15:0019:00 −1 CH4 1.4 0.7 0.6 0.7 Control 1.5 0.9 0.5 0.7 1 CH4 0.7 0.5 2.12.4 Control 0.5 0.2 1.5 1.8 2 CH4 2.4 1.0 1.5 1.2 Control 5.0 13.6 15.2  17.4  3 CH4 1.6 1.0 0.7 1.0 Control 7.8 4.5 3.5 2.5 7 CH4 1.8 1.61.0 1.2 Control 2.0 1.5 2.0 1.5 14 CH4 1.5 1.5 1.2 1.0 Control 1.8 2.01.2 2.0

In both trials dosing with CH4 led to a marked and significant (P<0.001for both trials) reduction in ruminal lactic acid concentration comparedto controls. In both trials control animals showed the expected sharpincrease in ruminal lactic acid concentration after abrupt addition ofreadily fermentable substrate to the rumen. In comparison ruminal lacticacid levels in CH4-treated animals remained more-or-less atpre-substrate-addition levels. This clearly suggested that the lacticacid being produced was largely utilized by CH4.

Evaluation of Isolate CH4 in High Producing Dairy Cows

The most relevant production data is presented in Tables 6 and 7. Datawas analyzed separately for all cows (15/treatment) and high producers(10/treatment), respectively.

TABLE 6 Effect of organism CH4 on productivity of lactating dairy cattlefrom calving to 80 days post partum (all cows). Contrast P< +CH4Treatment¹ vs 1 vs 3 vs Parameters 1 2 3 4 −CH4 2 4 Cows per treatment15 15 15 15 — — — Dry matter intake kg/d 24.6 24.1 23.1 22.2 0.28 0.590.32 Milk (kg/d) 36.4 34.0 33.8 32.2 0.10 0.16 0.34 Fat (%) 3.27 3.293.57 3.23 0.17 0.85 0.03 Protein (%) 3.10 3.10 3.14 3.07 0.43 0.93 0.23Body Weight 662 608 618 612 0.02 0.004 0.73 Condition score 2.80 2.482.45 2.28 0.06 0.08 0.36 Treatment 1: Control diet, 70% concentrate +CH4 Treatment 2: Control diet, 70% concentrate − CH4 Treatment 3:Control diet, 60% concentrate + CH4 Treatment 4: Control diet, 60%concentrate − CH4

TABLE 7 Effect of organism CH4 on productivity of lactating dairy cattlefrom calving to 80 days post partum (high producers). Contrast P< +CH4Treatment¹ vs 1 vs 3 vs Parameters 1 2 3 4 −CH4 2 4 Cows per treatment10 10 10 10 — — — Dry matter intake kg/d 24.6 25.4 24.3 22.6 0.44 0.430.06 Milk (kg/d) 39.3 35.9 35.2 34.8 0.13 0.06 0.82 Fat (%) 3.23 3.243.56 3.21 0.20 0.91 0.06 Protein (%) 3.10 3.10 3.15 3.02 0.28 0.93 0.11Body Weight 644 597 623 625 0.11 0.02 0.90 Condition score 2.71 2.262.34 2.44 0.20 0.02 0.61Effects of Megasphaera elsdenii Supplementation on Animal Health andFeedlot Performance

The delivery of live CH4 bacteria per animal averaged 2×10¹¹ colonyforming units per dose per animal throughout the feedlot trials. Themost relevant results are presented in Tables 8 to 11. The period ofweeks three to five in the feedlot is normally considered the mostcritical in terms of dietary adaptation. During weeks one and two thediet still has a higher roughage content, which gradually decreases;Intake starts at a comparatively low level and builds up gradually. Itis only from week three onwards that the diet is at its lowest roughagelevel (and highest concentrate level) and intakes are rapidly gettinghigher. By the beginning of week six animals will normally be consideredas adapted to the diet. Week three to five is really the critical periodof adaptation of animals to the high-concentrate diet.

TABLE 8 Average daily feed intake (kg as-fed material per animal perday) for CH4-treatment vs control (no CH4 added), for various periods inthe feedlot. Feedlot - Treatment period Plus CH4 Control (no CH4)P-value s.e. Week 1-2 7.30 7.15 0.35 0.109 Week 3-5 10.18 10.02 0.300.103 Week 1-13 9.64 9.50 0.29 0.092

Overall feed intake was slightly (but not significantly) higher for CH4than for control. During week 3-5, for steers not receiving ionophore,CH4 had a significantly (P<0.05) higher intake than controls (10.56 vs10.12), whilst no effect of CH4 was observed for animals receivingionophore. Also during this period, for steers on the low-roughagediets, CH4 tended (P<0.15) to have higher intakes than controls (10.14vs 9.80), whilst no effect of CH4 was observed for animals on thehigh-roughage diets.

TABLE 9 Average daily gain (kg per animal per day) for CH4-treatment vscontrol (no CH4 added), for various periods in the feedlot. Feedlot -Treatment period Plus CH4 Control (no CH4) P-value s.e. Week 1-2 1.761.81 0.51 0.053 Week 3-5 2.09 1.97 0.04 0.040 Week 1-13 2.19 2.20 0.670.019

Overall average daily gain (ADG) during the critical period of week 3-5was significantly (P=0.04) higher for CH4 than control treatments.During week 1-2 for all animals that did not receive CH4, thelow-roughage treatment had a significantly (P<0.05) lower ADG than thehigh-roughage treatment (1.61 vs 2.02). However, for animals that didreceive CH4, ADG was not significantly lower on low-roughage as comparedto high-roughage diets (1.70 vs 1.83). During week 3-5, for animals notreceiving an ionophore, CH4 had a significantly (P<0.05) higher ADG thancontrol animals (2.15 vs 1.96).

TABLE 10 Feed conversion ratio (kg feed per kg gain) for CH4-treatmentvs control (no CH4 added), for various periods in the feedlot. Feedlot -Treatment period Plus CH4 Control (no CH4) P-value s.e. Week 1-2 4.254.12 0.43 0.111 Week 3-5 4.89 5.14 0.04 0.081 Week 1-13 5.06 5.02 0.350.027

Overall animals treated with CH4 had a significant (P=0.04) ca 5%improvement in feed conversion ratio (FCR) over control animals duringweek 3-5. During week 1-2 for all animals that did not receive CH4, thelow-roughage treatment had a significantly (P=0.06) higher (lessdesireable) FCR than the high-roughage treatment (4.42 vs 3.83).However, for animals that did receive CH4, FCR was not significantlyhigher (less desireable) on low-roughage as compared to high-roughagediets (4.24 vs 4.25).

TABLE 11 Number of times (including multiple pulls of the same animal)and number of animals (multiple pulls of the same animal count only asone) that animals were pulled and treated for acidosis and bloat.Treatment +CH4 Contrl (−CH4) TOTAL: Total Roughage - 6 21 27 number Lowof pulls Roughage - 6 4 10 (incidents) Hi TOTAL: 12 25 37 TotalRoughage - 4 15 19 number Low of pulls Roughage - 5 2 7 (incidents) HiTOTAL: 9 17 26

For the CH4 treatments only half the number of animals suffered fromacidosis symptoms (one or more times) as compared to the controls. Thesame trend was observed if the total numbers of acidosis incidents wasconsidered. It is also clear that acidosis was much more prevalent onthe low-roughage diet and that CH4 treatment alleviated the problem onthe low-roughage diet.

Identification of Isolates using Phylogenetics, Based on 16S rRNA GeneSequences.

Comparative sequencing results showed that our isolates, which arerepresentatives of a larger phenotypically homogenous group, are between97 and 99% similar (Table 12). Table 13 outlines positions of signaturenucleotides suitable to distinguish the two recent Megasphaera elsdeniiisolates and the ATCC strains from each other. Insertions and deletionsaccounted for 22% of nucleotide differences between the four strains.The major nucleotide sequence differences between the strains occur atnucleotide positions 529-536 and 1105-1120 (Table 13). The high sequencesimilarity displayed between the different M. elsdenii strains is alsoconsistent with their similar phenotypic characteristics (Wiederhold,1994), which is furthermore reflected by their tight phylogeneticclustering. Strains of the species M. elsdenii share only 91 to 92%sequence similarity with M. cerevisiae, and the two species formdistinct clusters in the phylogenetic tree. The M. elsdenii clusterbifurcates into two monophyletic groups that evolved from the sameancestral taxonomic unit (ATU). The ATU from which M. cerevisiaeevolved, however, predates the one from which the M. elsdenii clusterevolved. The short branch lengths between M. elsdenii strains (OTU's)and their respective ATU's also indicate that they have evolved morerecently than the more deeply branched M. cerevisiae.

TABLE 12 Sequence similarity matrix of 16S rDNA sequences of M. elsdeniiand M. cerevisiae. ATCC 17752 ATCC25940 CH7 CH4 % G + C M. cerevisiae92.0 92.0 91.5 91.5 54.3 ATCC 25940 99.0 98.5 98.1 54.4 CH4 98.2 98.199.0 54.9 CH7 97.7 54.8 ATCC 17752 53.1 Sequence similarity values arebased on a comparison of a total of 1388 unambiguously alignednucleotide positions. The % G + C refer only to the respective aligned16S rDNA sequences.

TABLE 13 Sequence signatures defining different M. elsdenii isolates andstrains Nucleotides Position ^(a) ATCC 25940 ^(b) CH4 CH7 ATCC 17752  87G G A G 105 C T C T 170 T C C T 221 T C C T 241 G A A G 283 A G G A 418A * * * 529-530 CG * * CG 533-536 CG ** CGAC CGAC GC ** 539 T C C T550-552 TAC CGT CGT TAT 556 G A A G 711 G G G C 718 * * * G 850 A A G A1084  A G A A 1105-1108 TGGA AGGG AGGG TGGA 1117-1120 TCCA CCCT CCCTTCCA 1290 A * A * 1297-1300 AAGT CGGC AAGT CGGC 1396  A C A A 1425  A AG A 1437  G A C G 1492  T C C T ^(a) E. coli numbering system (Brosiuset al., 1978). ^(b) denotes the type strain. An asterisk indicates wherea gap was introduced during the alignment as a result of the occurrenceof a nucleotide deletion or insertion at any one position of thesequences of the respective isolates and strains.

The maximum likelihood method, which involves finding a tree, whichgives the highest probability of giving rise to the observed sequencedata (Felsenstein, 1981), was used to infer a phylogenetic tree from thesequences included in the alignment profile (FIG. 3). This method hasthe advantage above traditional parsimony methods, which could lead toinference of erroneous trees if different lineages evolve at unequalrates, in that it allows for evolutionary rates to differ betweendifferent lineages (Felsenstein, 1981). Since tree topology is alsoaffected by the number of organisms used and the selection of theoutgroups (Stackebrandt and Ludwig, 1994; Stackebrandt and Rainey,1995), a number of apparently related and apparently unrelatedorganisms, occurring in the rumen was included in an alignment profile.This was subsequently used to construct the tree. Although thephylogenetic tree was inferred from only nearly complete (92%) 16S rRNAgene sequences, which could reduce resolution between very closelyrelated organisms (Utaker et al., 1995; Li and Graur, 1991), the generaltopology of trees derived from either complete or partial sequences hasbeen shown to be in overall agreement with each other (Van Camp et al.,1993, Vandamme et al., 1996).

Vandamme et al. (1996) proposed that different isolates should beregarded members of the same species if they share more than 97% rRNAsequence homology, show phenotypic consistency and exhibit a significantdegree of DNA: DNA hybridization. Although the relationship between DNAsimilarity and 16S rRNA sequence homology between organisms is anythingbut linear, Fox et al. (1992) proposed that effective 16S rRNA sequenceidentity should imply that two organisms are members of the samespecies, since it would almost certainly be validated by the DNA: DNAhybridization. Although 16S rRNA sequence data alone may not besufficient in all cases to define a species, it is extremely useful indetermining to which species a strain probably belongs, once therelevant species is represented in a 16S rRNA sequence data base.Strains with almost identical 16S rRNA sequences should be assigned tothe same “rRNA superspecies” or “rRNA species complex”. It would thus beappropriate to assign isolates CH4 and CH7, phenotypically presumptivelyidentified M. elsdenii strains, to the same rRNA species complex, whichwould include reference strains ATCC 25940, the type strain of thespecies, and ATCC 17752. Since the phylogenetic relationships of therespective isolates are furthermore consistent with their phenotypiccharacteristics, these isolates can be considered strains of the speciesMegasphaera elsdenii. The fact that M. cerevisiae and M. elsdenii shareonly 92% 16S rDNA sequence homology, confirms, together with genotypicand phenotypic data, the division of the genus into two well resolvedspecies.

Of the rumen bacteria included in this study the ones which appear to bemost closely related to the Megasphaera cluster are Selenomonasruminantium and Quinella ovalis. The apparent phylogenetic relationshipbetween Megasphaera elsdenii and Selenomonas ruminantium is consistentwith some phenotypic and genotypic characteristics which the two speciesshare, such as similar DNA G+C content (53-54%), anaerobic nature,chemoorganotrophic metabolism and utilization of a similar range ofsubstrates (Stackebrandt et al., 1985; Stewart and Bryant, 1988;Haikara, 1992). The work of Stackebrandt et al. (1985), who made use ofthe oligonucleotide cataloguing technique for phylogenetic inferencebetween the species, supports this phylogenetic relationship.Selenomonas ruminantium on the other hand is also closely related to therelatively unknown Quinella ovalis, an organism that proliferates in therumen when sugar rich diets are fed to the animal. These organisms,although not established in culture yet, share some physiologicalcharacteristics with the large selenomonads found in sheep (Stewart andBryant, 1988). As expected, the most distant relatives of Megasphaerathat occur in the rumen are those contained in the archaeal methanogencluster, the members of which are believed to have appearedapproximately 600 to 800 million years ago (van Soest, 1994; Woese,1987). The evolutionary rates of these organisms are also slower thanthat of the Bacteria, and the primitiveness of the group is clearlyreflected by the deeply branched methanogen cluster.

The recent divergence of the different M. elsdenii strains couldpossibly be attributed to a refinement of its phenotype in order toadapt to the highly selective conditions in the rumen. According toWoese (1987), the evolution of the phenotype of an organism is a processduring which new or more efficient traits are gained in order to survivein its particular niche. Refinement would result in the organisms beingmetabolically versatile, as is the case with Megasphaera. The slowerevolving methanogens on the other hand are metabolically monotonous bycomparison.

This study has demonstrated the suitability of 16S rDNA sequencing todistinguish between closely related strains of the species M. elsdenii.Furthermore, it provided a phylogenetic framework for identification ofrecently isolated strains that have been characterized phenotypically.The framework would be of particular value in serving as a basis for thedesign of species and strain-specific probes intended for rumenecological studies.

CONCLUSIONS

Isolation

The incorporation of bromocresol purple to IRFL medium to facilitatedetection of lactate-utilizing bacteria proved to be successful in thecase of the faster-growing lactate-utilizes, which were of primeinterest in this study. In the early stages of incubation these producedpurple zones, concentric with the colonies, which contrasted clearlywith the yellowish background of the agar medium. However, on extendedincubation the pH gradient surrounding the colonies dissipated due todiffusion of ions and the whole background became purple.Differentiation between colonies of slower-growing lactate-utilizes andthose of organisms growing on other carbon sources present in the rumenfluid supplement then became difficult. M. elsdenii is not the dominantlactate-utilizing species in animals on high-concentrate diets (Mackieet al., 1978; Mackie & Gilchrist, 1979; Mackie et al., 1984; vanGylswyk, 1990), but there are a number of reasons why they may havepredominated in the selection and screening procedures.

Some of the colonies screened consisted of Selenomonads and othermorphological types. Most of these colonies were not chosen as there wasnot a positive indication that lactate could be utilized in the presenceof soluble sugars. Russell & Baldwin (1978) showed that M. elsdenii B159used glucose, maltose and lactate, but not sucrose simultaneously in amulti-substrate medium. Marounek et al. (1989) showed that for fourstrains of M. elsdenii lactate was used more rapidly than glucose inmedia with both carbon sources.

Certain other laboratories, which have studied the possibility ofinoculating ruminants on high-concentrate diets with lactate-utilizingorganisms to prevent an accumulation of lactate, have also worked withM. elsdenii strains (Das, 1979; Leedle et al., 1991; Robinson et al.,1992; Kung & Hession, 1995; Wiryawan & Brooker, 1995). It was notpossible to compare the growth rates of the AW and CH isolates tostrains in the literature to determine if they had faster growth ratesand if they were more acid tolerant, as no results were available inliterature. The AW and CH isolates can however be compared to the typestrain, M. elsdenii ATCC 25940.

For the AW isolates the range of pH values at which growth wasdetermined was not sufficient to determine the pH range for the isolatesor the optimum pH for growth. However, it can be assumed that theoptimum would be above pH 5.7 and the lowest pH would lie between pH 4.5and pH 4.9 for all but four of the isolates, as there was no growth onIRFL plates at pH 4.5. This agrees with work done on the type strain ofM. elsdenii ATCC 25940. The pH range for the type strain of M. elsdeniiATCC 25940 is pH 4.6 to 7.8 with the optimum for growth at pH 6.05(Therion et al., 1982).

For the CH isolates the optimum pH for growth is between pH 5 and 6. Inthe range of pH values tested, the highest growth rates were found at pH5.5. Both sets of isolates had higher growth rates on SDL medium thanthe type strain. The growth rates obtained for M. elsdenii ATCC 25940 inSDL medium is comparable to that obtained by earlier workers on lactatemedium (Therion et al., 1982).

The growth rates of the isolates on lactate were higher than on glucoseand maltose. This is in agreement with a previous study on M. elsdeniiATCC 25940, where growth rates on lactate medium between pH 5.0 and pH6.5 was found to be higher than in glucose medium (Therion et al.,1982). Outside this pH range growth rates on glucose were higher. Astudy on substrate preference in rumen bacteria reported that growth ofM. elsdenii B159 on lactate was slower than on glucose and maltose,however the pH of the media in the study was above pH 6.5, being between6.75 and 6.9 (Russell & Baldwin, 1978).

The composition of fermentation end products on lactate medium has beendetermined for four strains of M. elsdenii, including the type strainLC1 or ATCC 25940 (Marounek et al, 1989). These results showedstrain-to-strain variability in the proportions of fatty acids formed.Three of the strains produced little or no valeric acid while 22 mol %of the end-products of M. elsdenii L8 was valeric acid (Marounek et al.,1989). The nine AW isolates tested in the present study did not exhibitas much strain-to-strain variability as was the case in the strainstested by Marounek et al. (1989), but are similar to M. elsdenii L8,which was isolated from the rumen of a calf on a milk diet, as valericacid was produced. CH4, however, produced the same fermentationend-products as the type strain.

From the point of view of maximum biomass output rate in SDL mediumstrain AW15 would be the organism of choice for larger scale productionof cells for animal experiments with CH4 and AW01 being next in line.The time required to produce 100 g dry mass of cells on SDL medium in achemostat of 51 working volume would be 1.9 days for AW15 and 2.1 daysfor CH4 and AW01.

The growth rates of the selected isolates on lactate are high comparedto the type strain of M. elsdenii. The isolates are acid tolerant andcan grow at pH values below 5.0. They are resistant to ionophores,commonly added to feedlot diets, and can utilize both isomers of lacticacid even in the presence of glucose and maltose. The fermentation endproducts from lactate are VFA, which are an important energy source forthe ruminant. Propionate production is especially important in thefeedlot industry, as propionate is the main source of glucose for theruminant tissues. The isolates, therefore, have the characteristicsrequired for an effective product to combat lactic acidosis inruminants.

Cultivation of the lactate utilizes was successful using a medium thatdid not contain any rumen fluid. The only modification to the originalmedium was the increase in the vitamin content and the addition of yeastextract to the medium. Bacteria survived remarkably well on this mediumat 4° C. for up to 20 days, when used as working cultures.

The technique of using a pH-auxostat for the enrichment oflactate-utilizing rumen bacteria, with a predetermined combination ofbiochemical/physiological attributes, which would make them potentiallyhighly suitable for preventing and combating lactic acidosis in feedlotanimals was very successful. In most cases a fast-growing,morphologically homogeneous population became established in thefermenter within two days after the start of a run. Subsequent tests onthe cultures that were isolated from the fermenter contents by plating,confirmed that the cultures possessed the desired combination ofcharacteristics.

Presumptive identification of the isolates from the enrichments showedthat all but one belonged to the species Megasphaera elsdenii.

TABLE 14 A comparison between the pH-auxostat isolation technique and aconventional spread plate screening technique. Conventional Parameterspread plate Auxostat Time elapsed (days) 90 9 Man hours spent 180 7Sample: bacterial load (cfu) 12 × 10¹⁰ 14 × 10¹² Maximum specific growthrate (h⁻¹) 0.91 0.90 Biomass yield (g · l⁻¹) 0.60 0.59 Biomass outputrate g(l · h)⁻¹ 0.39 0.39

Since the isolates performed better than ATCC 25940 at pH values below6.0, they would be more suited to the ruminal pH encountered by feedlotanimals that usually is below pH 6.0. Furthermore, CH4 proved to be thebest suited isolate for trial experimentation on feedlot animals.

Cultivation of the lactate utilizing isolates on glucose proved not tobe a proposition due to the slow growth rates obtained compared to thegrowth rates obtained on lactate.

The thirty cows that were dosed with organism CH4 produced significantlymore milk (P=0.10), had a higher average body weight (P=0.02) and bodycondition score (P=0.06). The milk fat percentage of cows receiving the60% concentrate diet +CH4 were also significantly increased (3.57% vs.3.23%).

Dairy Animal Trials

Dry matter intakes did not differ but milk production was significantlyincreased by 3.4 kg/d from 35.9 to 39.3 kg/d (P=0.06) when cows were fedthe 70% concentrate diet and were dosed with organism CH4. Milkproduction tended to be increased (P=0.13) when all dosed cows werecompared to all non-dosed cows. Body weight and condition score wereincreased (P=0.02) when high producing cows receiving the highconcentrate diet were dosed with organism CH4. The dosing of cowsreceiving the 60% concentrate diet resulted in a significant increase inmilk fat percentage (P=0.06) with a tendency towards increased milkprotein (P=0.11). Milk components play an important role in current milkpayment schemes.

Dosing cows with organism CH4 significantly increased milk productionand positively affected milk composition, body weight and body conditionscore. Dry matter intakes were not affected; therefore results suggestthat dosing of cows with organism CH4 caused a more favorable rumenenvironment, which resulted in improved utilization of nutrients.

Feedlot Animal Trials

The significant improvement in average daily gain (ADG) and feedconversion ratio (FCR) during the critical period in the feedlot (week3-5), as well as the overall decrease in digestive disturbances, forCH4-treated animals as compared to controls, show that treatment offeedlot animals with CH4 can be effective in the following ways:

-   -   It aided the adaptation from roughage to concentrate diets, as        inherent in this experiment. CH4 also alleviated the poorer        performance of the low-roughage diet as compared to the        high-roughage diet during the early adaptation stages.    -   The application method used here for CH4 was effective in        allowing its expression as intended.    -   The use of CH4 was effective in preventing acidosis, as        evidenced directly by less observed cases of acidosis and        indirectly by improved performance during the critical feedlot        phase when acidosis is most likely to occur. All this was        observed on diets and dietary regimes with an exceptionally high        risk of acidosis (especially in the early feedlot stages), and        was more pronounced where no ionophore was used.    -   CH4 may be used as a veterinary agent to help in the prevention        and/or treatment of acidosis, as evidenced by the sharp decline        in acidosis cases for CH4 as compared to controls.    -   CH4 may be used to improve feedlot performance including growth        rate (and by implication to decrease time required for        finishing) and feed conversion efficiency.    -   CH4 can be used to allow the feeding of higher-concentrate        diets, i.e. the use of less roughage, and to increase the rate        of change from high roughage to high concentrate diets, i.e.        also the use of less roughage. This is further supported by the        observations, during the early feedlot phases, that the negative        effect of low-roughage diets, as compared to the high-roughage        diets, was largely alleviated when CH4 was added.

The applicants have further found that, in comparison with the knownstrains of M. elsdenii, the M. elsdenii CH4 strain is:

-   -   highly active and adapted to proliferate in the rumen of animals        on high-concentrate diets;    -   capable of proliferating at relatively low pH values below pH        5.0 and as low as 4.5, characterized as acute acidosis;    -   resistant to ionophore antibiotics commonly added to feedlot        diets; and    -   capable of preferentially using lactate as a carbon source even        in the presence of soluble carbohydrates such as glucose and        maltose.

Further advantages of this strain are that it:

-   -   has a relatively high growth rate, i.e. more than 0.938 h⁻¹;    -   has the ability to grow on reducing sugars as well as on        lactate;    -   has a relatively high biomass output rate, i.e. more than 0.39 g        (l.h)⁻¹;    -   is ionophore resistant;    -   produces predominantly acetate and not predominantly propionate        and butyrate; and    -   has a unique 16S rRNA sequence and is therefore a new strain.

The applicants have yet further found that animals challenged withmaltose, fed directly into the rumen, or abruptly changed from aroughage to a high-concentrate diet, produced no measurable build-up oflactate in the rumen when inoculated with CH4.

Furthermore, high producing dairy cows inoculated with CH4 has a 2.4 to3.2 liters higher production of milk, than control animals notinoculated with CH4. The body condition score as well as body weight ofthe inoculated cows were statistically significantly higher than thecontrol animals.

It will be appreciated that variations in detail are possible with amicroorganism according to the invention and its uses without departingfrom the scope of the appended claims.

REFERENCES

-   1. Abdo, K. M. & Cahilly, G. M. (1974). Ruminant feed additive and    method of preparing the same. U.S. Pat. No. 3,857,971, 1-30.-   2. Allison, M. J., Bucklin, J. A. & Dougherty, R. W. (1964). Ruminal    changes after overfeeding with wheat and the effect of intraruminal    inoculation on adaptation to a ration containing wheat. J Anim Sci    23, 1164-1170.-   3. Braun, U., Rihs, T. & Schefer, U. (1992). Ruminal lactic acidosis    in sheep and goats. Veterinary Record 130, 343-349.-   4. Das, N. K. (1979). Ruminant feed additive. U.S. Pat. No.    4,138,498, 1-14.-   5. Dawson, K. A. & Allison, M. J. (1988). Digestive disorders and    nutritional toxicity. In The rumen microbial ecosystem, pp. 445-459.    Edited by P. N. Hobson. London: Elsevier Applied Science.-   6. Gray, W. M. (1978). Microbial interactions in defined continuous    culture systems effecting anaerobic cellulose degradation, PhD    thesis: Clemson University.-   7. Hession, A. O. & Kung, L., Jr. (1992). Altering rumen    fermentation by microbial inoculation with lactate-utilizing    microorganisms. J Anim Sci 70, 311. (Abstract)-   8. Jannasch, H. W., 1977. Growth kinetics of aquatic bacteria. In:    Aquatic Microbiology. Skinner, F. A. & Shewan, J. M. (Eds), Academic    Press, London, 55-57.-   9. Kung, L., Jr. & Hession, A. O. (1995). Preventing in vitro    lactate accumulation in ruminal fermentations by inoculation with    Megasphaera elsdenii. J Anim Sci 73, 250-256.-   10. Krieg, R., 1981. Enrichment and isolation. In: Manual of Methods    for General Bacteriology. Gerhardt, P. (Ed.), American Society for    Microbiology, Washington, 112-114.-   11. Lederberg, J. & Lederberg, E. M. (1952). Replica plating and    indirect selection of bacterial mutants, J Bact 63, 399-406.-   12. Leedle, J. A. Z., Greening, R. C. & Smolenski, W. J. (1991).    Ruminal bacterium for preventing lactic acidosis. International    Application No : PCT/US91/00857 1-41.-   13. Mackie, R. I., Gilchrist F. M. C., Robberts, A. M.,    Hannah, P. E. & Schwartz, H. M. (1978). Microbiological and chemical    changes in the rumen during the stepwise adaptation of sheep to high    concentrate diets. J agric Sci, Camb 90, 241-242.-   14. Mackie, R. I., Gilchrist, F. M. C. & Heath, S. (1984). An in    vivo study of ruminal micro-organisms influencing lactate turnover    and its contribution to volatile fatty acid production. J agric Sci,    Camb 103, 37-51.-   15. Mackie, R. I. & Gilchrist, F. M. C. (1979). Changes in    lactate-producing and lactate-utilizing bacteria in relation to pH    in the rumen of sheep during stepwise adaptation to a    high-concentrate diet Appl Environ Microbiol 38, 422-430.-   16. Mackie, R. I. & Heath. S. (1979). Enumeration and isolation of    lactate-utilizing bacteria from the rumen of sheep. Appl Environ    Microbiol 38, 416-421.-   17. Marounek, M., Fliegrova, K. & Bartos, S. (1989). Metabolism and    some characteristics of ruminal strains of Megasphaera elsdenii.    Appl Environ Microbiol 55, 1570-1573.-   18. Olumeyan, D. B., Nagaraja, T. G., Miller, G. W., Frey, R. A. &    Boyer, J. E. (1986). Rumen microbial changes in cattle fed diets    with or without salinomycin. Appl Environ Microbiol 51, 340-345.-   19. PIRT, S. J., 1975. Principles of Microbe and Cell Cultivation.    Blackwell Scientific Publications, Oxford, 41-210.-   20. Pryce, (1969). A modification of the Barker-Summerson method for    the determination of lactic acid. Analyst 94, 1151.-   21. Robinson, J. A., Smolenski, W. J., Greening, R. C., Ogilvie, M.    L., Bell, R. L., Barsuhn, K. & Peters, J. P. (1992). Prevention of    acute acidosis and enhancement of feed intake in the bovine by    Megasphaera elsdenii 407A J Anim Sci 70, 310. (Abstract)-   22. Rogosa, M. (1984). Anaerobic Gram-negative cocci. In Bergey's    manual of systematic bacteriology Volume 1, pp. 680-685. Edited    by N. R. Krieg & J. G. Holt, Baltimore/London: Williams & Wilkins.-   23. Russell, J. B. & Baldwin, R. L. (1978). Substrate preferences in    rumen bacteria: evidence of catabolite regulatory mechanisms. Appl    Environ Microbiol 36, 319-329.-   24. Slyter, L. L. (1976). Influence of acidosis on rumen function. J    Anim Sci 43, 910-929.-   25. Stewart, C. S. & Bryant, M. P. (1988). The rumen bacteria. In    The rumen microbial ecosystem, pp. 21-75. Edited by P. N. Hobson.    London: Elsevier Applied Science.-   26. Therion, J. J., Kistner, A & Kornelius, J. H. (1982). Effect of    pH on growth rates of rumen amylolytic and lactilytic bacteria. Appl    Environ Microbiol 44, 428-434.-   27. Van Gylswyk, N. O. (1990). Enumeration and presumptive    identification of some functional groups of bacteria in the rumen of    dairy cows fed grass silage-based diets. FEMS Microbiol Ecol 73,    243-254.-   28. Veldkamp, H. 1970. Enrichment cultures of prokaryotic organisms.    In: Methods in Microbiology, Vol. 3A, Norris, J. R. & Ribbons, D. W.    (Eds), Academic Press, London, 305-361.-   29. Wilker, B. L., Paege, L. M. & Baker, E. (1971). Bacterial    culture for facilitating dietary adaptation of ruminants. Patent    Office, London 1251483, 1-13.-   30. Wiryawan, K G. & Brooker, J. D. (1995). Probiotic control of    lactate accumulation in acutely grained sheep. Aust J Agric Res 46,    1555-1568.    Cow Experiments-   31. DAWSON, J. A. 1995. The use of yeast strain 8417 in manipulating    ruminant high concentrate diets. Proc. 56th Minnesota Nutr. Conf. &    Alltech Inc. Technical Symposium. Sep. 18-20, Bloomington, Minn.-   32. DONOVAN, J. 1997. Subacute acidosis is costing no millions.    Hoards Dairyman, Sep. 25 p. 666.-   33. HALL, M. B. 1999. Management strategies against ruminal    acidosis. Proc. Florida Ruminant Nutr. Symp. Univ. Florida,    Gainesville, Fla.-   34. HUTJENS, M. F. 1995. Feeding applications for the high producing    cow. Cornell Nutr. Conf. Oct. 24-26. Dept Anim. Sci., Cornell Univ.,    Ithaca, N.Y.-   35. HUTJENS, M. F. 1999. How and when feed additives may or may not    pay, Hoards, Dairy man, Sep. 25, 1999.-   36. KELLY, E. R. & LEAVER, J. D. 1990. Lameness in dairy cattle and    the type of concentrates given. Anim. Prod. 51: 221.-   37. KUNG, L. 2000. Direct fed microbials for dairy cows. Proc.    Florida Ruminant Nutr. Symp. Univ. Florida, Gainesville, Fla.-   38. KUNG, L & HESSION, O. A. 1995. Altering rumen fermentation by    microbial inoculation with lactate utilizing micro-organisms. J.    Anim. Sci. 73:250.-   39. MANSON, R. J. & LEAVER, J. D. 1988. The influence of concentrate    amount on locomotion and clinical lameness in dairy cattle. Anim.    Prod. 47-185.-   40. McDANIEL, B. T. & WILK, J. C. 1989. Lameness in dairy cattle.    Proc. Southwest Nutr. Mgmt, Conf., Feb. 2-3, Dept Anim. Sci., Univ.    Arizona, Tucson, Ariz.-   41. NOCEK, J. E. 1995. Energy metabolism and rumen acidosis. Proc.    Tri-state Dairy Nutr. Conf., May 23-24. Indiana Univ., Fort Wayne,    Ind.-   42. NOCEK, H. E. 1997. Bovine acidosis. Implications on    laminitis. J. Dairy Sci 80:1005.-   43. NORDLUND, K. V. 1995. Questions and answers regarding    rumenocentesis and the diagnosis of herd based sub-acute rumen    acidosis. Proc. 4-State Applied Nutr. Conf., Aug. 2-3, Univ.    Wisconsin, Extension, Madison, Wis.-   44. NORDLUND, K. V., GARRET, E. F. & OETZEL, G. R. 1995. Herd based    rumenocentesis: A clinical approach to the diagnosis of subacute    rumen acidosis. The Compendium Food Animal, Aug. 1995, p. 48.-   45. OETZEL, G. R. & SMITH, R. A. 2000. Clinical aspects of ruminal    acidosis in dairy cattle. Proc. 33^(rd). Conf. American Assoc. Bov.    Pract, Rapid City, S.D. Sep. 21-23.-   46. OWENS, F. N., SECRIST, D., HILL, J. & GILL, D. 1996. A new look    at acidosis. Proc. Southwest Nutr. Conf., Feb. 1, Phoenix, Ariz.-   47, VOGEL, G. J. & PARROT, C. 1994. Mortality survey in feedyards:    The incidence of death from digestive, respiratory and other causes    in feedyards on the great plains. The Compendium Feb. 1994: Food    Animal, p. 227.-   48. ROBINSON, J. A., SMOLENSKI, W. J., GREENING, R. C., OGILVIE, R.    L., BELL, R. L., BARSUHN, K & PETERS, J. P. 1992 Prevention of acute    acidosis and enhancement of feed intake in the bovine by Megasphaera    elsdenii 407A. J. Anim Sci. (Suppl. 1): 310 (Abstr)    Phylogenetics-   49. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. A.    Smith, J. G. Seidman, and K. Struhl. 1987. Current Protocols in    Molecular Biology. Vol 1 and 2. John Wiley and Sons, New York,-   50. Brosius, J., M. L. Palmer, P. J. Kennedy and H. F. Noller. 1978.    Complete nucleotide sequence of the 16S ribosomal RNA gene from E.    coli. Proc. Natl. Acad. Sci. 75:4801-4805.-   51. Dorsch, M. and E. Stackebrandt. 1992 Some modifications in the    procedure of direct sequencing of PCR amplified 16S rDNA. J.    Microbiol. Methods. 16:271-279.-   52. Elsden, S. R. and D. Lewis. 1953. The production of fatty acids    by a gram-negative coccus. Biochem. J. 55:183-189.-   53. Elsden, S. R., B. E. Volcani, F. M. C. Gilchrist and D.    Lewis. 1956. Properties of a fatty acid forming organism isolated    from the rumen of sheep. J. Bacteriol. 72:681-689.-   54. Engelmann, U. and N. Weis. 1985. Megasphaera cerevisiae sp.    nov.: A new Gram-negative obligately anaerobic coccus isolated from    spoiled beer. Syst Appl. Microbiol. 6:287-290.-   55. Felsenstein, J. 1981. Evolutionary trees from DNA sequences: a    maximum likelihood approach. J. Mol. Evol. 17:368-376.-   56. Fox, G. E., J. D. Wisotzkey and P. Jurtshuk, Jr. 1992 How close    is close: 16S rRNA sequence identity may not be sufficient to    guarantee species identity. Int. J. Syst Bacteriol. 42:166-170.-   57. *Genetics Computer Group. 1991. Program manual for the GCG    package, version 7. Madison, Wis., USA-   58. Gutierrez, J., R. E. Davis, I. L. Lindahl and E. J.    Warwick. 1959. Bacterial changes in the rumen during the onset of    feed-lot bloat of cattle and characteristics of Peptostreptococcus    elsdenii n. sp, Appl. Microbiol. 7:16-22.-   59. Haikara, A 1992. The genera Pectinatus and Megasphaera, p.    1993-2004. In A. Barlows, H. Trooper, M. Dworkin, W. Harder and    K.-H. Schleifer (ed), The prokaryotes. A handbook on the biology of    bacteria: ecophysiology, isolation, identification, application, 2nd    ed., vol ψψ. Springer Verlag, New York.-   60. Hutson, R. A., D. E. Thompson, and M. D. Collins. 1993. Generic    interrelationships of saccharolytic Clostidium botulinum types B, E    and F and related clostridia as revealed by small-subunit rRNA gene    sequences. FEMS Microbiol. Lett. 108:103-110.-   61. Lane, D. J., B. Pace, G. J. Olsen, D. A. Stahl, M. L. Sogin,    and N. R. Pace. 1985. Rapid determination of 16S ribosomal RNA    sequences for phylogenetic analyses. Proc. Natl. Acad. Sci. USA    82:6955-6959.-   62. Li, W. -H., and D. Graur. 1991. Fundamentals of molecular    evolution. Sunderland, Mass. Sinauer Associates Inc.-   63. Maidak, B. L., Olsen, G. J., Larsen, N., Overbeek, R.,    McCaughey, M. J. and Woese, C. R. 1996. The ribosomal database    project (RDP). Nucleic Acids Res 24:82-85.-   64. Olsen, G. J., H. Matsuda, R. Hagstrom, and R. Overbeek. 1994.    FastDNAml—a tool for construction of phylogenetic trees of DNA    sequences using maximum likelihood. CABIOS 10:41-48.-   65. Rogosa, M. 1971. Transfer of Peptostreptococcus elsdenii    Gutierrez et al. to a new genus, Megasphaera [M. elsdenii (Gutierrez    et al.) comb. nov]. Int. J. Syst Bacteriol. 21:187-189.-   66. *Sambrook, J., Fritsch, E. F. and Maniatis, T. 1989. Molecular    cloning. A laboratory manual. Second edition (Eds. Ford, N.,    Nolan, C. and Ferguson, M.). Cold Spring Harbor, Laboratory Press.-   67. Smith, S. 1992 Genetic Data Environment, version 2.0:    Documentation. Harvard University & University of Illinois.-   68. Stackebrandt E., and W. Ludwig. 1994. The importance of choosing    outgroup reference organisms in phylogenetic studies: the Atopobium    case. Syst Appl. Microbiol. 17:39-43.-   69. *Stackebrandt, E., and F. A. Rainey. 1995. Partial and complete    16S rDNA sequences, their use in generation of 16S rDNA phylogenetic    trees and their implications in molecular ecological studies.    Molecular Microbial Ecology Manual 3.1.1:1-17. Kuwer Academic    Publishers, Netherlands.-   70. Stackebrandt, E., H. Pohla, R. Kroppenstedt, H. Hippe, and C. R.    Woese. 1985. 16S rRNA analyses of Sporomusa, Selenomonas, and    Megasphaera: on the phylogenetic origin of Gram-positive Eubacteria.    Arch. Microbiol. 143:270-276.-   71. Stackebrandt, E., and O. Charfreitag. 1990. Partial 16S rRNA    primary structure of five Actinomyces species: phylogenetic    implications and development of an Actinomyces isrealii-specific    oligonucleotide probe. J. Gen. Microbiol. 136:37-43.-   72. Stewart C. S., and M. P. Bryant 1988. The rumen bacteria. p.    21-75. In Hobson, P. N. (ed), The rumen microbial ecosystem.,    Elsevier Applied Science, London-   73. Sugihara, P. T., V. L. Sutter, H. R., Attebery, K. S. Brichnell,    and S. M. Finegold. 1974. Isolation of Acidominococcus fermentans    and Megasphaera elsdenii from normal human feces. Appl. Microbiol.    27:274-275.-   74. Utaker, J. B., L. Bakken, Q. Q. Jiang, and I. F. Nes. 1995.    Phylogenetic analysis of seven new isolates of ammonia-oxidizing    bacteria based on 16S rRNA gene sequences. Syst Appl. Microbiol.    18:549-559.-   75. Van Camp, G., Y. Van De Peer, S. Nicolai, J.-M. Neefs, P.    Vandamme, and R. De. Wachter. 1993. Structure of 16S and 23S    ribosomal RNA genes in Campylobacter species: phylogenetic analysis    of the genus Campylobacter and presence of internal transcribed    spacers. Syst Appl. Microbiol. 16:361-368.-   76. Vandamme, P., B. Pot M. Gillis, P. De Vos, K. Kersters, and J.    Swings. 1996. Polyphasic taxonomy, a consensus approach to bacterial    systematics. Microbiol. Rev. 60:407-438.-   77. van Soest, P. J. 1994. Nutritional ecology of the ruminant    2^(nd) Edition. Comstock Publishing Associates, Ithaca.-   78. Wiederhold, A. H. 1994. Isolation, selection and cultivation of    lactic acid-utilizing rumen bacteria for the treatment of chronic    and acute acidosis. MSc. thesis. University of Natal,    Pietermaritzburg.-   79. Woese, C. R. 1987. Bacterial evolution. Microbiol. Rev.    51:221-271.

The invention claimed is:
 1. A biologically pure bacterial culture of anacid tolerant strain of Megasphaera elsdenii selected from the groupconsisting of the M. elsdenii strain CH3 deposited at NCIMB, Aberdeen,Scotland, UK under number NCIMB 41787, and the M. elsdenii strain CH7deposited at NCIMB, Aberdeen, Scotland, UK under number NCIMB 41788,wherein the acid-tolerant strain has a maximum growth rate of at leastabout 0.5 h⁻¹ on lactate medium at pH 5.0.
 2. The biologically purebacterial culture of claim 1 which is further characterised by itsability to utilize lactate in the presence of a sugar selected from thegroup consisting of glucose and maltose.
 3. A composition forfacilitating the adaptation of ruminants from a roughage-based diet to ahigh-energy concentrate-based diet, the composition comprising thebacterial culture of claim
 1. 4. A method for facilitating theadaptation of ruminants from a roughage-based diet to a high-energyconcentrate-based diet, the method comprising administering to the rumenof said ruminants an effective amount of the composition of claim
 3. 5.A feed-additive for ruminants comprising a carrier and an effectiveamount of the bacterial culture of claim
 1. 6. The feed-additiveaccording to claim 5 wherein the culture is disposed in an anaerobiccontainer.
 7. A method for the treatment of ruminal lactic acidosiscomprising anaerobically administering to the rumen of a ruminant aneffective amount of a bacterial culture according to claim
 1. 8. Themethod of claim 7 wherein the method treats at least one ruininal lacticacidosis, rumenitis, ruminal lactic acidosis induced laminitis, ruminallactic acidosis induced bloat and liver abscesses.
 9. A composition forthe treatment of ruminal lactic acidosis comprising an effective amountof a bacterial culture according to claim
 1. 10. The composition ofclaim 9, wherein the method treats at least one of ruminal lacticacidosis, rumenitis, ruminal lactic acidosis induced laminitis, ruminallactic acidosis induced bloat and liver abscesses in ruminants.
 11. Apreparation for the treatment of ruminal lactic acidosis comprising aninoculum of a bacterial culture according to claim 1 and a sterileanaerobic growth medium.
 12. The preparation of claim 11 wherein theculture and the medium are disposed in separate chambers of an anaerobiccontainer, wherein the chambers are anaerobically connectable to eachother.
 13. The preparation of claim 12, wherein the preparation treatsat least one of ruminal lactic acidosis, rumenitis, ruminal lacticacidosis induced laminitis, ruminal acidosis induced bloat and liverabscesses in ruminants.
 14. A method of achieving in a ruminant at leastone of increased milk production; improved growth rate; lower incidenceof lactic acidosis and related diseases; in feeds; and, the methodcomprising administering to the rumen of a ruminant an effective amountof the bacterial culture of claim
 1. 15. A method according to claim 14wherein the culture is administered anaerobically.
 16. The biologicallypure bacterial culture of claim 1, which is further characterised by itsresistance to ionophores.
 17. The biologically pure bacterial culture ofclaim 1, which is further characterised by its capability to producepredominantly acetate.
 18. The biologically pure bacterial culture ofclaim 1 further characterised by a maximum growth rate of at least about0.36 h⁻¹ on lactate medium at about pH 4.75.
 19. The biologically purebacterial culture of claim 1 wherein the acid-tolerant strain of Melsdeni has the identifying characteristics of a growth rate on lactatemedium at pH 4.5 of at least 0.1 h⁻¹ and a maximum growth on lactatemedium at pH 5.5 of at least about 0.86 h⁻¹.